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The Soft Agar Colony Formation Assay
Published on: October 27, 2014
ADAMTS8 inhibited lung cancer progression through suppressing VEGFA
Yutian Zhang1, Kang Hu2, Ziyi Qu1
1Graduate School, Tianjin University of Traditional Chinese Medicine, Tianjin, PR China.
Background:
ADAMTS8 expression has been identified to be low in many cancers including lung cancer. However, the specific functions and regulatory system of ADAMTS8 remain to be unveiled.
Purpose:
To study the potential modulatory mechanism of ADAMTS8 in lung cancer in cell and xenograft mice models.
Methods:
Differential expression of ADAMTS8 in lung cancer was analyzed on online tools. So was the overall survival curve in association with ADAMTS8/VEGFA expression in lung cancer patients. RT-qPCR was applied to validate the ADAMTS8 expression in lung cancer cell lines H460 and A549, with the normal lung epithelial cell Beas-2b as a control. Thereafter, overexpressed and knockdown plasmids were constructed for transfection. Colony and flow cytometry methods were used for cell proliferation and apoptosis. RT-qPCR and Western blot methods validated the changes in VEGFA after ADAMTS8 regulation in cells. Tube formation and Transwell methods were applied to observe the changes in tube formation and migration in HUVECs induced by tumor conditioned medium (TCM). Stable-transfected cells were injected subcutaneously into nude mice. H&E and Immunohistochemistry were applied to analyze the pathological differences and protein changes of ADAMTS8, VEGFA and CD31.
Results:
High ADAMTS8 was correlated with high overall survival rate in lung cancer patients. ADAMTS8 was also abnormally downregulated in NSCLC cells. Upregulation of ADAMTS8 suppressed cell proliferation and enhanced apoptosis while downregulation of ADAMTS8 promoted cell proliferation and decreased apoptosis. VEGFA was negatively correlated with ADAMTS8 in lung cancer tissues. Upregulation of ADAMTS8 inhibited VEGFA in mRNA and protein levels. Further, knockdown of ADAMTS8 induced tube formation and migration of HUVECs and upregulation of ADAMTS8 inhibited this. In addition, upregulation of ADAMTS8 in nude mice inhibited tumor growth and also suppressed VEGFA and CD31 in tumors.
Conclusion:
ADAMTS8 inhibited lung cancer progression through suppressing VEGFA in lung cancer.
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